Contribution of factor H-Binding protein sequence to the cross-reactivity of meningococcal native outer membrane vesicle vaccines with over-expressed fHbp variant group 1

Factor H-binding protein (fHbp) is an important meningococcal vaccine antigen. Native outer membrane vesicles with over-expressed fHbp (NOMV OE fHbp) have been shown to induce antibodies with broader functional activity than recombinant fHbp (rfHbp). Improved understanding of this broad coverage would facilitate rational vaccine design. We performed a pair-wise analysis of 48 surface-exposed amino acids involved in interacting with factor H, among 383 fHbp variant group 1 sequences. We generated isogenic NOMV-producing meningococcal strains from an African serogroup W isolate, each over-expressing one of four fHbp variant group 1 sequences (ID 1, 5, 9, or 74), including those most common among invasive African meningococcal isolates. Mice were immunised with each NOMV, and sera tested for IgG levels against each of the rfHbp ID and for ability to kill a panel of heterologous meningococcal isolates. At the fH-binding site, ID pairs differed by a maximum of 13 (27%) amino acids. ID 9 shared an amino acid sequence common to 83 ID types. The selected ID types differed by up to 6 amino acids, in the fH-binding site. All NOMV and rfHbp induced high IgG levels against each rfHbp. Serum killing from mice immunised with rfHbp was generally less efficient and more restricted compared to NOMV, which induced antibodies that killed most meningococci tested, with decreased stringency for ID type differences. Breadth of killing was mostly due to anti-fHbp antibodies, with some restriction according to ID type sequence differences. Nevertheless, under our experimental conditions, no relationship between antibody cross-reactivity and variation fH-binding site sequence was identified. NOMV over-expressing different fHbp IDs belonging to variant group 1 induce antibodies with fine specificities against fHbp, and ability to kill broadly meningococci expressing heterologous fHbp IDs. The work reinforces that meningococcal NOMV with OE fHbp is a promising vaccine strategy, and provides a basis for rational selection of antigen sequence types for over-expression on NOMV

Peripheral blood cellular profile at pre-lymphodepletion is associated with CD19-targeted CAR-T cell-associated neurotoxicity

BackgroundInfusion of second generation autologous CD19-targeted chimeric antigen receptor (CAR) T cells in patients with R/R relapsed/refractory B-cell lymphoma (BCL) is affected by inflammatory complications, such as Immune Effector Cell-Associated Neurotoxicity Syndrome (ICANS). Current literature suggests that the immune profile prior to CAR-T infusion modifies the chance to develop ICANS.MethodsThis is a monocenter prospective study on 53 patients receiving approved CAR T-cell products (29 axi-cel, 24 tisa-cel) for R/R-BCL. Clinical, biochemical, and hematological variables were analyzed at the time of pre-lymphodepletion (pre-LD). In a subset of 21 patients whose fresh peripheral blood sample was available, we performed cytofluorimetric analysis of leukocytes and extracellular vesicles (EVs). Moreover, we assessed a panel of soluble plasma biomarkers (IL-6/IL-10/GDF-15/IL-15/CXCL9/NfL) and microRNAs (miR-146a-5p, miR-21-5p, miR-126-3p, miR-150-5p) which are associated with senescence and inflammation.ResultsMultivariate analysis at the pre-LD time-point in the entire cohort (n=53) showed that a lower percentage of CD3+CD8+ lymphocytes (38.6% vs 46.8%, OR=0.937 [95% CI: 0.882-0.996], p=0.035) and higher levels of serum C-reactive protein (CRP, 4.52 mg/dl vs 1.00 mg/dl, OR=7.133 [95% CI: 1.796-28], p=0.005) are associated with ICANS. In the pre-LD samples of 21 patients, a significant increase in the percentage of CD8+CD45RA+CD57+ senescent cells (median % value: 16.50% vs 9.10%, p=0.009) and monocytic-myeloid derived suppressor cells (M-MDSC, median % value: 4.4 vs 1.8, p=0.020) was found in ICANS patients. These latter also showed increased levels of EVs carrying CD14+ and CD45+ myeloid markers, of the myeloid chemokine CXCL-9, as well of the MDSC-secreted cytokine IL-10. Notably, the serum levels of circulating neurofilament light chain, a marker of neuroaxonal injury, were positively correlated with the levels of senescent CD8+ T cells, M-MDSC, IL-10 and CXCL-9. No variation in the levels of the selected miRNAs was observed between ICANS and no-ICANS patients.DiscussionOur data support the notion that pre-CAR-T systemic inflammation is associated with ICANS. Higher proportion of senescence CD8+ T cells and M-MDSC correlate with early signs of neuroaxonal injury at pre-LD time-point, suggesting that ICANS may be the final event of a process that begins before CAR-T infusion, consequence to patient clinical history

Real World Evidence of CAR T-Cell Therapies for the Treatment of Relapsed/Refractory B-Cell Non-Hodgkin Lymphoma: A Monocentric Experience

Large B-cell lymphomas (LBCL) are the most common types of non-Hodgkin lymphoma. Although outcomes have improved thanks to the introduction of rituximab-based chemoimmunotherapy, certain LBCL still represents a challenge because of initial resistance to therapy or recurrent relapses. Axicabtagene ciloleucel (axi-cel) and tisagenlecleucel (tisa-cel) are second-generation autologous CD19-targeted chimeric antigen receptor (CAR) T-cell therapies approved for patients with relapsed/refractory (R/R) LBCL, based on the results of phase II pivotal single-arm trials ZUMA-1 (for axi-cel) and JULIET (for tisa-cel). Here, we report patients outcomes with axi-cel and tisa-cel in the standard of care (SoC) setting for R/R LBCL, treated at our Institution. Data were collected from patients who underwent leukapheresis between August 2019 and February 2021. Toxicities were graded and managed according to the institution’s guidelines. Responses were assessed as per Lugano 2014 classification. Of the 30 patients who underwent leukapheresis, 18 (60%) received axi-cel, while 12 (40%) tisa-cel. Grade 3 or higher cytokine release syndrome and neurotoxicity occurred in 10% and 16% patients, respectively. Best objective and complete response rates were 73.3% and 40%, respectively. Treatment in SoC setting with CD19 CAR T-cell therapies for R/R LBCL showed a manageable safety profile and high objective response rate

List of NOMV used in this study.

<p>List of NOMV used in this study.</p

Characteristics of <i>N</i>. <i>meningitidis</i> strains used in in this study.

<p>Characteristics of <i>N</i>. <i>meningitidis</i> strains used in in this study.</p

NOMV size characterisation by HPLC-SEC/MALS and dls.

<p>NOMV size characterisation by HPLC-SEC/MALS and dls.</p

Effects of sRNA deletions on meningococcal infection.

<p>(A) Northern blots experiments validating the deletion of the indicated sRNAs. RNA was extracted from 2996 wild type (WT) and relative deletion strains grown until stationary phase for all sRNAs, except for Bns1/1563-1564_F for which the growth condition was minimal medium supplemented with glucose. 16s rRNA is provided as a loading control. (B) Competitive index (CI) from infant rats infected with WT <i>N</i>. <i>meningitidis</i> 2996 and the indicated sRNA KO strains at a 1:1 ratio. Solid circles indicate individual animals. Horizontal solid and dashed black lines indicate means and medians for each group, respectively. A horizontal dotted line is set to CI = 1, indicating no effect on meningococcal infection due to deletion of the respective sRNAs. The number of animals in each group and the results of statistical analysis are shown above the graph (2-tails, unpaired T-test analysis were performed to assess the statistical significance for each group of CIs to be different from 1; * = pval ≤ 0.05, ns = no statistical significance). (C) Table summarizing result of CI index assay with statistical analysis.</p

Serum bactericidal responses induced in individual mice immunised with NOMV or recombinant fHbp.

<p>Mice were immunised twice with NOMV (A-E), or with recombinant fHbp (F-I), four weeks apart, as described in the materials and methods. Serum samples obtained two weeks after the second dose were tested against a set of seven strains, expressing the four different fHbp ID types: ID 1 (), ID 5 (), ID 9 (), or ID 74 (). Each symbol represents the reciprocal titre of an individual mouse; horizontal bars represent geometric mean titres of the group.</p

Global Transcriptome Analysis Reveals Small RNAs Affecting <i>Neisseria meningitidis</i> Bacteremia

<div><p>Most bacterial small RNAs (sRNAs) are post-transcriptional regulators involved in adaptive responses, controlling gene expression by modulating translation or stability of their target mRNAs often in concert with the RNA chaperone Hfq. <i>Neisseria meningitides</i>, the leading cause of bacterial meningitis, is able to adapt to different host niches during human infection. However, only a few sRNAs and their functions have been fully described to date. Recently, transcriptional expression profiling of <i>N</i>. <i>meningitides</i> in human blood <i>ex vivo</i> revealed 91 differentially expressed putative sRNAs. Here we expanded this analysis by performing a global transcriptome study after exposure of <i>N</i>. <i>meningitides</i> to physiologically relevant stress signals (e.g. heat shock, oxidative stress, iron and carbon source limitation). and we identified putative sRNAs that were differentially expressed <i>in vitro</i>. A set of 98 putative sRNAs was obtained by analyzing transcriptome data and 8 new sRNAs were validated, both by Northern blot and by primer extension techniques. Deletion of selected sRNAs caused attenuation of <i>N</i>. <i>meningitides</i> infection in the <i>in vivo</i> infant rat model, leading to the identification of the first sRNAs influencing meningococcal bacteremia. Further analysis indicated that one of the sRNAs affecting bacteremia responded to carbon source availability through repression by a GntR-like transcriptional regulator. Both the sRNA and the GntR-like regulator are implicated in the control of gene expression from a common network involved in energy metabolism.</p></div

Validation of the high density data through NrrF and AniS expression profile.

<p>Expression profile of the NrrF (A) and AniS (B) sRNAs. Graphical representations of tilling array results during growth in the indicated conditions. The <i>y</i> axis shows the M value (log2ratio of expression of the experimental condition versus the reference), while on the <i>x</i> axis the genomic position of the probes is depicted in relationship to the schematic representation of the locus under the graph. ORFs and sRNAs are represented as white or grey horizontal arrows, respectively and the transcriptional starting sites and the terminators of sRNAs are indicated. Vertical black dashed lines indicate the position of sRNAs. Dotted black horizontal lines limit the background signal in which the probes are not considered to be regulated. Each dot in the graph corresponds to the average of the M values for one probe from three independent replicates in the indicated condition. On the upper right, experimental validation of the tilling array results by Northern blot (NrrF) or primer extension (AniS). 16s ribosomal RNAs provided a loading control for the Northern Blot experiment. The only notable inconsistency among microarray data and validation is the down-regulation of AniS in primer extension experiments during heat shock treatment. This discrepancy could be due to the fact that microarray probes and the primer used for validation do not recognize the same portion of AniS.</p